Upregulated ZFAS1 expression and increased ferroptosis involved in diabetic cardiomyopathy and HG-treated cardiomyocytes.
The differentially HF-related genes profiles in diabetic patients and non-diabetic patients were screened out from the GSE26887 in the GEO database, lncRNA-ZFAS1 was significantly upregulated in the diabetic patients with HF (Fig. 1A). To determine whether ZFAS1 was really involved in diabetic cardiomyopathy, the expression of ZFAS1 in the left ventricular myocardial tissues of db/db mice and HG-treated cardiomyocytes was determined by RT-qPCR. The result revealed that ZFAS1 was significantly upregulated at the same way (Fig. 1B-C).
As shown in Fig. 1D, FTH1, a key iron storage protein involved in iron metabolism and act as ferritinophagy biomarkers, was decreased in the left ventricular myocardial tissues of db/db mice evaluated by immunohistochemical staining (Fig. 1D). 4-hydroxynonenal (4-HNE), the final product of lipid hydroperoxidation, was increased in the left ventricular myocardial tissues of db/db mice evaluated by immunohistochemical staining (Fig. 1D). Glutathione peroxidase 4 (GPX4), which could terminate the process of ferroptosis, was decreased in the left ventricular myocardial tissues of db/db mice measured by western blot (Fig. 1E-F). In keeping with in vivo results, FTH1 was seen colocalized rarely in the cytoplasm in HG-treated cardiomyocytes (Fig. 1G). We also observed a reduction in the expression of GPX4 in HG-treated cardiomyocytes measured by western blot (Fig. 1H-I). Taking together, we found that the expression of ZFAS1 was upregulated and ferroptosis was increased in diabetic cardiomyopathy and HG-treated cardiomyocytes.
Inhibition of ZFAS1 repressed ferroptosis in diabetic cardiomyopathy and HG-Treated Cardiomyocytes.
To further identify the function of ZFAS1 in the process of DCM, we injected Ad-ZFAS1, Ad-sh-ZFAS1 into the left ventricle free wall of mice. Masson staining showed a significant decrease of collagen deposition in the left ventricular myocardial tissues of db/db + Ad-ZFAS1 group (Fig. 2A). Inhibition of ZFAS1 could restore the expression of FTH1, reduce the expression of 4-HNE evaluated by immunohistochemical staining (Fig. 2B), rescue the expression of GPX4 and inhibit the expression of apoptosis-related genes including Cleaved caspase 3, Bax, and Bcl-2 measured by western blot (Fig. 2C-D). Inhibition of ZFAS1 in HG-treated cardiomyocytes could increase intracellular GSH levels assessed by MBB staining to a certain extent (Fig. 2E), restore the distribution of FTH1 in the cytoplasm (Fig. 2F), alleviate the mitochondrial membrane potential as revealed by the transition from red fluorescence to green fluorescence measured by JC-1 staining (Fig. 2G), rescue the expression of GPX4 and inhibit the expression of apoptosis-related genes including Cleaved caspase 3, Bax, and Bcl-2 measured by western blot (Fig. 2H-I). Altogether, these results suggested that inhibition of ZFAS1 could prevent ferroptosis from diabetic cardiomyopathy and HG-treated cardiomyocytes.
lncRNA-ZFAS1 can bind with miR-150-5p to regulate expression of CCND2.
The differentially role of micro-RNAs profiles pathophysiology of diabetic cardiomyopathy were screened out from the GSE44179 in the GEO database, we found that miR-150-5p was substantial reduced (Fig. 3A). To determine whether miR-150-5p was really involved in diabetic cardiomyopathy, the expression of miR-150-5p in the left ventricular myocardial tissues of db/db mice and HG-treated cardiomyocytes was determined by RT-qPCR. The result revealed that miR-150-5p was significantly downregulated at the same way (Fig. 3B-C).
To elucidate the potential molecular mechanism by which ZFAS1 and miR-150-5p regulated DM, we attempted to explore the underlying target bind site between ZFAS1 and miR-150-5p. The predicted bind site of miR-150-5p and ZFAS1 was displayed in the Fig. 3D analyzed by bioinformatic program TargetScan. Dual luciferase reporter assay demonstrated that transfection with miR-150-5p mimics significantly reduced the relative firefly luciferase activity of wt-ZFAS1 whereas the mut-ZFAS1 luciferase activity was not affected (Fig. 3E). Further, we compared the sequences of ZFAS1 with that of miR-150-5p analyzed by the bioinformatics program RNAhybrid and investigated that ZFAS1 contains a binding site of miR-150-5p (Fig. 3F). What’s more, we performed a biotin-avidin pull-down assay to explore whether miR-150-5p could directly bind to ZFAS1. ZFAS1 was pulled down by biotinylated wild-type miR-150-5p, the inability of miR-150-5p to pull down ZFAS1 when introduction of miR-150-5p mutations that destroy base paring between ZFAS1 and miR-150-5p, indicating that the identification of miR-150-5p to ZFAS1 is sequence specific (Fig. 3G). We also performed inverse pull-down assay to test if ZFAS1 could pull-down miR-150-5p, the results showed that miR-150-5p could be co-precipitated by ZFAS1using a biotin-labeled-specific ZFAS1 probe (Fig. 3H).
The GSE44179 from GEO database presented us that CCND2 was significantly decreased in rat ventricles after STZ injection (Fig. 3I). To determine whether CCND2 was really involved in diabetic cardiomyopathy, the expression of CCND2 in the left ventricular myocardial tissues of db/db mice and HG-treated cardiomyocytes was determined by western blot. The result revealed that CCND2 was significantly downregulated in the same way (Fig. 3J-M). The bioinformatic program TargetScan provided information for the predicted binding site of miR-150-5p and CCND2 (Fig. 3N). Additionally, the dual-luciferase reporter assay demonstrated that transfection with miR-150-5p mimics significantly reduced the relative firefly luciferase activity of wt-CCND2 whereas the mut-CCND2 luciferase activity was not affected (Fig. 3O). These results supported that ZFAS1 can bind with miR-150-5p to regulate the expression of CCND2.
ZFAS1 promoted ferroptosis in diabetic cardiomyopathy and HG-treated Cardiomyocytes through modulating miR-150-5p.
Because of the interaction between ZFAS1 and miR-150-5p, we further explored whether ZFAS1 was able to regulate ferroptosis through miR-150-5p. As shown in Fig. 4A, stimulation of miR-150-5p significantly decreased collagen deposition in the left ventricular myocardial tissues of db/db mice which was similar to the function of ZFAS1 inhibition. However, stimulation of ZFAS1 definitely abolished the positive effect of miR-150-5p detected by Masson staining. Overexpression of miR-150-5p could restore the expression of FTH1, reduce the expression of 4-HNE evaluated by immunohistochemical staining as the function of inhibition of ZFAS1, whereas overexpression of ZFAS1 offset the positive effect of miR-150-5p (Fig. 4B). Overexpression of miR-150-5p could rescue the expression of GPX4 and CCND2 and inhibit the expression of apoptosis-related genes including Cleaved caspase 3, Bax, and Bcl-2 measured by western blot which were similar to the function of ZFAS1, whereas overexpression of ZFAS1 counteracted the positive effect of miR-150-5p (Fig. 4C-D).
Subsequently, we explored whether ZFAS1 regulate ferroptosis through miR-150-5p in HG-treated cardiomyocytes. As shown in Fig. 4E, HG-treated cardiomyocytes transfected with Ad-sh-ZFAS1 or mimic miR-150-5p all made sense in the increase of intracellular GSH levels assessed by MBB staining (Fig. 4E), whereas overexpression of ZFAS1 counteracted the positive effect of miR-150-5p. Moreover, Ad-ZFAS1 administration significantly reversed the effect of miR-150-5p, as demonstrated by a reduction in the expression of FTH1 as detected via immunofluorescence (Fig. 4F), the decrease in mitochondrial membrane potential as detected via JC-1 staining (Fig. 4G), a reduction in the expression of GPX4 and CCND2 and a reduction in the expression of apoptosis-related genes including Cleaved caspase 3, Bax, and Bcl-2 measured by western blot (Fig. 4H-I).
Taking together, these results indicated that inhibition of ZFAS1 could suppress ferroptosis in diabetic cardiomyopathy and HG-treated Cardiomyocytes through targeting miR-150-5p. lncRNA-ZFAS1 acted as a ceRNA to sponge miR-150-5p.
ZFAS1 promoted ferroptosis in diabetic cardiomyopathy and HG-treated Cardiomyocytes through modulating CCND2.
To further elucidate the role of CCND2 in the positive function of ZFAS1 inhibition against ferroptosis in diabetic cardiomyopathy and HG-treated Cardiomyocytes, the left ventricular myocardial tissues of db/db mice were injected with Ad-sh-ZFAS1, Ad-ZFAS1, Ad-CCND2, or Ad-sh-CCND2. Interestingly, overexpression of CCND2 exerted a marked effect in inhibition of ferroptosis likewise the function as the inhibition of ZFAS1, however, these effects were eliminated when stimulation of ZFAS1 corroborated by the increased collagen deposition measured by Masson staining (Fig. 5A), the decreased expression of FTH1 and increased the expression of 4-HNE measured by immunohistochemical staining (Fig. 5B), the decreased expression of GPX4 and the increased expression of apoptosis-related genes including Cleaved caspase 3, Bax, and Bcl-2 measured by western blot (Fig. 5C-D) in the left ventricular myocardial tissues of db/db mice, the decreased of intracellular GSH levels assessed by MBB staining(Fig. 5E), the reduction expression of FTH1 as detected via immunofluorescence (Fig. 5F), the decreased mitochondrial membrane potential as detected via JC-1 staining (Fig. 5G), the reduction in the expression of GPX4 and the reduction in the expression of apoptosis-related genes including Cleaved caspase 3, Bax, and Bcl-2 measured by western blot (Fig. 5H-I). Cumulatively, these results demonstrated that ZFAS1 inhibition suppressed ferroptosis in diabetic cardiomyopathy and HG-treated Cardiomyocytes through modulating CCND2. lncRNA-ZFAS1 acted as a ceRNA to sponge miR-150-5p can regulate CCND2.